Acquisition of a Leica Stellaris 8 Point Confocal Laser Scanning Microscope

The Center for Advanced Bioimaging (CAB) Denmark is a core facility center at the University of Copenhagen, aiming to provide researchers with advanced, state-of-the-art imaging equipment, as well as consulting in data acquisition and analysis.

The center contributes to a great range of basic as well as applied research projects, both via our own and our network’s expertise and by enabling researchers to use the high-end equipment available at the center.

A state-of-the-art point confocal microscope takes a central position in research requiring microscopic imaging, among a broad variety of disciplines. The current confocal microscopes at CAB are the most booked instruments and often bottlenecks due to the high demand and increasing down-times due to service and maintenance. At the same time, technology has rapidly developed since the time of their acquisition, and the need for renewal at the facility is apparent. The new microscope will allow research otherwise not possible at or facility, e.g. by providing drastically improved speed, resolution and access to the lifetime domain of fluorescence microscopy. It will be fully upgradable and the center’s workhorse-instrument for many years.

The Stellaris system is the only system equipped with all the required features.

• most notably, the exclusive white light laser (WLL) allows for free tuning of the excitation wavelength over the broad spectral range from 440 nm to 790 nm. This is crucial for us, as the occurrence of autofluorescence in plant samples (which are a primary target of the research conducted at CAB), as well as the need to simultaneously image many fluorophores with often overlapping spectra, make fine optimisation of the used wavelengths necessary to avoid crosstalk between channels;

• at the same time, the pulsed WLL, together with the special HyD S and HyD X detectors and electronics, allow for acquisition of fluorescence lifetime data. This offers another way of separating/eliminating autofluorescence (one of our main priorities) via lifetime-gating;

• the freely tunable WLL goes hand-in-hand with freely tunable Acousto-Optical-Beam-Splitter (AOBS) and Acousto-Optical-Tunable-Filter (AOTF), making the system unique in its free tunability in both excitation and emission;

• besides the spectral flexibility, high speed and quantum efficiency are requirements, as the center’s focus is on imaging of biological and often living samples, with rapid physiological processes to be captured at high speeds and with minimal damage due to intense laser illumination;

• the Stellaris system is going to be an absolutely central element in CABs instrument park, and as such, the possibility of future upgrades is a must. Specifically, the ability to further extend the system’s fluorescence lifetime capabilities (FALCON module), add multi-photon excitation support (DIVE module), STED super resolution support and/or label-free Coherent Anti-Stokes Raman Scattering (CARS) support are a requirements.

Leica Microsystems CMS GmbH, has several patents in Germany and Europe that relate to essential components of company’s confocal laser scanning microscopes. The European patent EP 1 164 403 B1 is in force in Germany, France and Great Britain. The corresponding patent in America, US 6 710 918, is in force as well. These patents relate to a scan-microscope with a white light laser.

Compatibility with our existing microscopy/confocal equipment by Leica Microsystems is an additional advantage. There are some 200 active users of the confocal microscopes at CAB who are trained at Leica SP5 confocals. The combination of white-light excitation laser and prism-enabled spectrometric emission filtering is a unifying element between the existing confocals and the Stellaris system that will ease the switch between these systems.